Cells were lysed using Easy-Blue (Intron Biotechnology) and their RNA was purified according to the manufacturer’s instructions. Briefly, 2 μg RNA was heated to 70°C for 5 min and then subjected to reverse transcription for 2 h using M-MLV reverse transcriptase, oligo-dT, and random hexamer primers. Then, 1 μl of cDNA was used as a template for quantitative PCR. Target mRNA levels were normalized with the mRNA level of GAPDH. The qPCR primers used for the target genes were: (human) hGAPDH qF, 5′-CTTCGCTCTCTGCTCCTCCT-3′; hGAPDH qR, 5′-GTTAAAAGCAGCCCTGGTGA-3′; (human) hCTGF qF, 5′-AAAAGTGCATCCGTACTCCCA-3′; hCTGF qR, 5′-CCGTCGGTACATACTCCACAG-3′; (human) hCYR61 qF, 5′-CCAATGACAACGCCTCCTG-3′; hCYR61 qR, 5′-TGGTGCAGCCAGAAAGCTC-3′; (human) hAMOTL2 qF, 5′-GCATTGAGAAGCTGGAAAGC-3′; hAMOTL2 qR, 5′-CTTGTTCCGCATGGTCTTCT-3′; (human) hANKRD1 qF, 5′-AGTAGAGGAACTGGTCACTGG-3′; hANKRD1 qR, 5′-TGGGCTAGAAGTGTCTTCAGAT-3′.
Easy blue
Manufactured by iNtRON Biotechnology
Sourced in Cameroon
Easy-BLUE is a molecular biology reagent kit designed for the rapid and efficient extraction of DNA from various sample types, including plant, animal, and microbial samples. The kit utilizes a simple and streamlined protocol to isolate high-quality DNA suitable for downstream applications such as PCR, sequencing, and cloning.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (6 mentions)
Sensifast sybr no rox kit, by Meridian Bioscience (3 mentions)
Accupower rt premix, by Bioneer (3 mentions)
Sybr premix ex taq, by Takara Bio (3 mentions)
M mlv reverse transcriptase, by Enzynomics (3 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (3 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Rotor gene 6000 series software 1, by Qiagen (2 mentions)
Taqman one step master mix, by Thermo Fisher Scientific (2 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr kit, by Qiagen (2 mentions)
Rotor gene q, by Qiagen (2 mentions)
Revertra ace qpcr rt master mix, by Toyobo (2 mentions)
Cdna synthesis kit, by Toyobo (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Iq sybr green supermix, by Bio-Rad (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
M mulv reverse transcriptase, by New England Biolabs (1 mentions)
42 protocols using easy blue
1
Quantitative RT-PCR Protocol for Gene Expression
2
Apoptosis-Related Gene Expression Analysis
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A549 cells were treated with CG for 48 h, harvested, and lysed in 1 mL easy-BLUE™ (iNtRon Biotechnology, SungNam, Korea). The RNA was isolated in accordance with the manufacturer’s instructions, and cDNA was obtained by using M-MuL V reverse transcriptase (New England Biolabs, Beverly, MA, USA). Real-time qPCR was performed using a relative quantification protocol using Rotor-Gene 6000 series software 1.7 (Qiagen, Venlo, Netherlands) and a SensiFAST™ SYBR NO-ROX Kit (BIOLINE, London, UK). The expression of all target genes was normalized to that of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase, GAPDH. Each sample contained one of the following primer sets: Fas F: 5′-CGGACCCAGAATACCAAGTG-3′ and R: 5′-GCCACC CCAAGTTAGATCTG-3′; FasL F: 5′-GGGGATGTT TCAGCTCTTCC-3′ and R: 5′-GTGGCCTAT TTG CTT CTCCA-3′; DR5 F: 5′-CACCTTGTACACGATGC TGA-3′ and R: 5′-GCTCAACAA GTGGTCCTCAA-3′; FADD F: 5′-GGGGAAAGATTGGAGAAGGC-3′ and R: 5′-CAGTTCTCAGTGACTCCCG-3′; SOD2 F: 5′-TATAGAAAGCCGAGTGTTTCCC-3′ and R: 5′-GGGATGCCTTTCTAGTCC TATTC-3′; Catalase F: 5′-GGGATCTTTTAACGCCATT-3′ and R: 5′-CCAGTTTACCAA CTGGATG-3′; Thioredoxin F: 5′-GAAGCTCTG TTTGGTGCTTTG-3′ and R: 5′-CTCGAT CTGCTTCACCATCTT-3′; GAPDH F: 5′-GGCTG CTTTTAACTCTGGTA-3′ and R: 5′-TGG AAGATGGTGATGGGATT-3′.
3
Quantitative Real-Time PCR Gene Expression Analysis
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Total RNA was extracted using easy-BLUE (iNtRON Biotechnology, Dajeon, Korea) in accordance with the manufacturer’s protocol. cDNA was synthesized from 2 µg of total RNA using TOPscriptTM Drymix Kit (Enzynomics, Dajeon, Korea). qRT-PCR was performed using TB Green ® Premix Ex TaqTM II (Tli RNAse H Plus) (TaKaRa, Japan) with Applied Biosystems QuantStudio™ 1 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Gene expression data were normalized based on the use of the same amount of total RNA samples. The relative fold change of gene expression was calculated using the ∆Ct method. Sequences of primers are listed in Table 2 .
4
Quantifying Gene Expression in Hepatic Samples
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RNA was separated from each hepatic tissue sample using Easy Blue (iNtRON Biotechnology, Seongnam, Korea). TaqMan one-step master mix (Applied Biosystems, Waltham, MA, USA) was used for reverse transcriptase and real-time PCR of the mRNA samples. PCR was performed using an ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Fatty acid synthase (FAS) (Mm01204974_m1), SREBP1 (Mm00550338_m1), tumor necrosis factor (TNF) (Mm00443258_m1), interleukin-6 (IL-6) (Mm00446190_m1), and β-actin (Mm99999915_g1) levels were validated using TaqMan gene expression assays. Relative gene expression was calculated using StepOne software v2.3 (Applied Biosystems, Foster City, CA, USA) through relative threshold cycle (CT) values. β-actin was used as the reference value. Three repetitions were performed for the harvested cell sample and the hepatic sample of animals.
5
Splenic B Cell Transcriptional Analysis
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Splenic B cells were purified with CD19 mAb-conjugated microbeads (Miltenyi Biotech, Auburn, CA) according to the manufacturer’s instructions, to > 95% purity. Total RNA was isolated from sorted Splenic CD19+ B cells (1 × 107 cells) by using easy-BLUE (iNtRON Biotechnology, Gyeonggi, Korea) and reverse-transcribed with the ImProm-II reverse transcription first-strand synthesis system (Promega, Madison, WI) according to the manufacturer’s protocol. PCR was performed at 95°C for 2 min, 30 cycles of 95°C for 20 sec, 58°C for 40 sec, 72°C for 30 sec, and 72°C for 5 min. Primers used as follow: mouse BRD2 (forward 5′-CCACGAAAAGACTTGCC TGA-3′, reverse 5′-CAGCGTGCTTCTTTGAGAGC-3′); mouse BRD3 (forward 5′-CTATGCGTGGCCCTTTTACA-3′, reverse 5′-CTTCCTTTTGACCGTGCTGA-3′); mouse BRD4 (forward 5′-CAAAAGGAAGAGGACGAGGG-3′, reverse 5′-ACAGGTG GAGGAGGGTTCTG-3′); mouse IL-10 (forward 5′-GGCCCAG AAATCAAGGAGCA-3′, reverse 5′-GGGGGATGACAGTAGGG GAA-3′); mouse GAPDH forward 5′-TGACGTGCCGCCTGGA GAAA-3′, reverse 5′-AGTGTAGCCCAAGATGCCCTTCAG-3′.
6
Quantifying IP-10 mRNA Expression
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Total cellular RNA was isolated from a confluent monolayer of cells using easy-BLUE (iNtRON Biotechnology, Seongnam, Korea) according to the manufacturer's protocol. cDNA was synthesized from 1 μg of total RNA with reverse transcription, and PCR was performed using 3 μl of cDNA. The primer sequences were as follows. Human IP-10: forward, 5′-GTA CCT CTC TCT AGA ACC GTA CG-3′ and reverse, 5′-CCT TTA CAG ATT TTC TAG AG-3′ GAPDH: forward, 5′-GTC AGT GGT GGA CCT GAC CT-3′ and reverse, 5′-TGT GAG GAG GGG A GA TTC AG-3′. mRNA expression values were normalized to GAPDH mRNA levels for each experimental condition.
7
RNA Extraction and qPCR Analysis of MARCO
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Total RNA was extracted from BMDMs using easy-BLUE (Intron Biotechnology, Korea), according to the manufacturer's instructions. One microgram of total RNA was reverse transcribed to cDNA using ReverTra Ace qPCR RT Master Mix and cDNA Synthesis kit (Toyobo, Osaka, Japan). Equal amounts (1 μl) of cDNA were used for real-time PCR on a Rotor-Gene Q (Qiagen, Hilden, Germany) using a SYBR Green PCR kit (Qiagen). GAPDH was used for normalization. The following primers were used for real-time PCR: MARCO forward: 5′-ATCCTGCTCACGGCAGGTACT-3′; MARCO reverse: 5′-GCACATCTCTAGCATCTGGAGCT-3′; GAPDH forward: 5′-CAGTGGATGCAGGGATGATGTTCT-3′; GAPDH reverse: 5′-GTGGAGATTGTTGCCATCAACG-3′.
8
Quantifying AdipoR1, AdipoR2, E-cadherin, COX-2, and T-cadherin in HCT116 cells
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Total RNA was extracted from HCT116 cells using the easy-BLUE (iNtRON Biotechnology) total RNA extraction kit. The amount and purity of the extracted RNAs were determined by spectrophotometry. Complementary DNA samples were prepared by reverse transcription using 2 μg RNA per sample. qRT-PCR was carried out using the TaqMan Expression Master Mix (Applied Biosystems) for AdipoRs. The primers used were as follows: the FAM-labeled Adiponectin Receptor1 (assay ID Hs00360422_m1) was used for AdipoR1 and the FAM-labeled Adiponectin Recptor2 (assay ID Hs00226105_m1) was used for AdipoR2. qRT-PCR was carried out using the SYBR Green Master Mix (Applied Biosystems) for E-cadherin, COX-2, and T-cadherin. The primers used were: 5′-TGGGTTATTCCTCCCATCAG-3′ (foward) and 5′-TTTGTCAGGGAGCTCAGGAT-3′ (reverse) for E-cadherin, 5′-TTCAAATGAGATTGTGGAAAAAT-3′ (forward) and 5′- AGATCATCTCTGCCTGAGTATCTT-3′ (reverse) for COX-2 and 5′- GATGTTGGCAAGGTAGTCGAT-3′ (forward) and 5′-GCTCCCTGTGTTCTCATTGAT-3′ (reverse) for T-cadherin. Expression levels were presented as a ratio to GAPDH (assay ID Hs99999905_ml) as the internal control. qRT-PCR was performed using a Real-Time 7000 system (Applied Biosystems). A non-template control was included in each experiment. All patient samples and non-template controls were assayed in duplicate.
9
Osteogenic Differentiation Assay Protocol
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β-glycerophosphate, ascorbic acid, Alizarin red S stain and an ALP staining kit were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). BMP2 was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). The NE-PER Nuclear and Cytoplasmic Extraction Reagent was obtained from Pierce (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The easy-BLUE™ and StarTaq™ reagents were purchased from Intron Biotechnology, Inc. (Seongnam, Korea), and the AccuPower RT-PreMix was purchased from Bioneer Corporation (Daejeon, Korea). The miR isolation kit was purchased from Ambion (Thermo Fisher Scientific, Inc.). The Mir-X™ miRNA First-Strand Synthesis kit and SYBR Advantage miRNA qRT-PCR kit were obtained from Clontech Laboratories, Inc. (Mountainview, CA, USA). The polymerase chain reaction (PCR) primers were synthesized by Takara Korea Biomedical, Inc. (Seoul, Korea). SYBR Premix Ex Taq™ was purchased from Takara Bio, Inc. (Otsu, Japan). Dual-Glo luciferase assay kit was obtained from Promega Corporation (Madison, WI, USA). The concentration of PGE2 was detected using a Prostaglandin E2 ELISA kit (ab133021; Abcam, Cambridge, UK).
10
Quantitative Analysis of MMP-1 Expression
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Total RNA was isolated from cells using easy-BLUE (Intron Biotechnology, Daegeon, Republic of Korea). The polymerase chain reaction (PCR) conditions for MMP-1 and the housekeeping gene GAPDH were as follows: 35 cycles of 94°C for 45 sec, 52°C for 1 min, and 72°C for 1 min. Primer pairs (Bionics, Seoul, Republic of Korea) were as follows (forward and reverse, respectively): MMP-1, 5′-GGAGGAAATCTTGC-TCAT-3′ and 5′-CTCAGAAAGAGCAGCATC-3′; GAPDH, 5′-AA - GGTCGGAGTCAACGGATTT-3′ and 5′-GCAGTGAGGGTCTC- TC TCCT-3′. The amplified products were resolved by 1% agarose gel electrophoresis, stained with ethidium bromide, and photographed under UV illumination.
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